1. Volume 20, Issue 20, Pages 1787-1798 (October 2010)
A Direct Role for Cohesin in Gene Regulation and Ecdysone Response in Drosophila Salivary Glands 
Andrea Pauli, Joke G. van Bemmel, Raquel A. Oliveira, Takehiko Itoh, Katsuhiko Shirahige, Bas van Steensel, Kim Nasmyth 
Current Biology 
Volume 20, Issue 20, Pages (October 2010)
DOI: /j.cub
Copyright © 2010 Elsevier Ltd Terms and Conditions

2. Current Biology 2010 20, 1787-1798DOI: (10.1016/j.cub.2010.09.006)
Copyright © 2010 Elsevier Ltd Terms and Conditions

3. Figure 1
Cleavage of Cohesin Causes Major Transcriptional Changes in Salivary Glands
Transcriptional changes in salivary glands in the absence versus presence of cohesin were assessed after heat-shock-induced tobacco etch virus protease (TEV) cleavage of cohesin. Green indicates upregulation, red indicates downregulation in the absence of cohesin.
(A) Outline of the heat shock (hs)-TEV system used for differential gene expression profiling in salivary glands. Larvae carrying the hs-TEV construct and containing cohesin complexes with wild-type or TEV-cleavable (purple arrow) Rad21 were raised at 18°C before TEV protease was induced ubiquitously in late third-instar larvae by heat shock (45 min, 37°C). Salivary glands were dissected 10–12 hr after hs, followed by RNA isolation. Rad21 is shown in blue, TEV in purple (see also Figure S1A).
(B) Top: list of the 20 most downregulated and 20 most upregulated genes upon cohesin cleavage in salivary glands identified by microarray analysis (see also Figures S1B and S1C, Table S1, and Table S2). Genes are sorted in descending order based on their average fold change in transcript levels in the absence versus presence of cohesin across seven independent microarrays. A minus indicates fold downregulation. Genes at rank 50 are also given. Bottom: differential expression of selected candidates was confirmed by quantitative real-time PCR (qRT-PCR). Each bar represents the average fold change in the absence versus presence of cohesin of at least three independent experiments (error bars are standard deviations of the mean). Gene names are given above each bar; fold changes are given below/inside each bar.
(C) ChIP-CHIP analysis of the distribution of Pol-II (black plots) in Rad21 (+cohesin) and Rad21TEV (−cohesin) salivary glands 10–12 hr after heat-shock induction of TEV protease. Cohesin binding (blue plots) in salivary glands was assessed by DNA adenine methylase identification (DamID; Dam-Rad21). ChIP-CHIP data is represented as fold enrichment of IP over input (MAT scores; log scale; highly enriched regions (p < ) are in orange). DamID data are represented as the relative enrichment of methyl-adenine-marked DNA from Dam-Rad21 glands over Dam-only glands (log2 scale). EcR (downregulated, red box) and ush (upregulated, green box) loci are shown as representative examples (see also Figure S2 for further examples).
(D) Rad21-bound domains (cohesin domains) across a randomly chosen 2 Mb chromosomal region of chromosome 2L. Shown is the relative enrichment of DamRad21 versus Dam-only signal (log2 scale). Rad21 domains are highlighted as blue bars; genes are indicated as gray bars. The size distribution of the total number of 870 Rad21 domains in salivary glands is shown on the right.
(E) Differentially expressed genes are enriched in Rad21 binding. Shown are percentages of transcriptional start sites (TSSs) of cohesin-dependent genes (up- or downregulated) and of all genes that localized inside (blue), outside (gray), or at the border (white) of Rad21-bound regions. The asterisk (∗) indicates Fisher's exact test: p < 0.01.
(F) Average Rad21 binding at the TSSs of upregulated genes (green), downregulated genes (red), and all genes (gray).
Current Biology  , DOI: ( /j.cub )
Copyright © 2010 Elsevier Ltd Terms and Conditions

4. Figure 2 Cohesin Regulates the Expression of Ecdysone-Responsive Genes
Differential gene expression in salivary glands after cohesin cleavage was assessed at ecdysone-responsive genes [25].
(A) Percentages of ecdysone-responsive and of all genes that are upregulated (green), downregulated (red), and unchanged (white) after TEV cleavage of cohesin. The asterisk (∗) indicates Fisher's exact test: p < 0.01.
(B) Log2-fold changes in gene expression after TEV cleavage of cohesin for ecdysone-responsive genes (yellow) versus all genes (gray).
(C) Pol-II- and Rad21-binding profiles at Eip74EF and Eip75B are shown as representative examples for ecdysone-responsive loci (see Figure 1C for further details; see also Figure S2).
(D) Ecdysone-responsive genes that are differentially expressed upon cohesin cleavage are enriched in Rad21 binding. Shown are percentages of TSSs of ecdysone-responsive cohesin-dependent genes, of all ecdysone-responsive genes, and of all genes that localized inside (blue), outside (gray), or at the border (white) of Rad21-bound regions. The asterisk (∗) indicates Fisher's exact test: p < 0.01.
(E) Average Rad21 binding at the TSSs of four different categories of genes: ecdysone-responsive genes that are upregulated (green) or downregulated (red) after cohesin cleavage, all ecdysone-reponsive genes (yellow), and all genes (gray).
Current Biology  , DOI: ( /j.cub )
Copyright © 2010 Elsevier Ltd Terms and Conditions

5. Figure 3 Timed Salivary Gland-Specific Cleavage of Cohesin
(Aa–Ac) Outline of the tubGal80ts/SG-TEV system that enables control of timing and salivary gland specificity of cohesin cleavage. Larvae surviving on Rad21TEV and encoding tubGal80ts and SG-TEV (F4-Gal4, UAST-NLS-TEV) were raised at 18°C, the permissive temperature for the ubiquitously expressed Gal4 inhibitor Gal80ts. By shifting late third-instar larvae to the restrictive temperature (30°C), Gal80ts is degraded, which enables the salivary gland-specific Gal4 driver F4-Gal4 to induce TEV protease expression specifically in salivary glands (Aa). (Ab) and (Ac) summarize the states of transcription and cohesin, respectively, in salivary glands at 18°C and 30°C. Rad21 is shown in blue, TEV in purple, Gal80ts in orange.
(B) Schematic comparison of the effects of the hs-TEV versus SG-TEV/tubGal80ts systems on the level of functional cohesin complexes (presence of uncleaved Rad21TEV) after induction of TEV protease. Concentrations of TEV (purple) and Rad21TEV (blue) are plotted against time. Rad21TEV starts to reaccumulate in the hs-TEV system about 15 hr after TEV induction because of degradation of TEV and resynthesis of Rad21TEV (top), whereas continuous expression of TEV in salivary glands at 30°C prevents reappearance of full-length Rad21TEV in this tissue in the SG-TEV/tubGal80ts system (bottom).
(C) Western blot analysis of salivary gland extracts from SG-TEV/tubGal80ts larvae surviving either on Rad21TEV or Rad21. Extracts were prepared before (t = −1; TEV off) and at different time points after shifting third-instar larvae to 30°C (TEV on). Blots were probed with the indicated antibodies. Full-length Rad21 (∗) and the C-terminal TEV cleavage fragment (∗∗), as well as full-length Histone H3 (<) and N-terminally clipped Histone H3 (< <), are indicated. Tubulin and Ponceau stainings were used as loading controls.
(D) Representative polytene chromosome spreads from SG-TEV/tubGal80ts crawling third-instar larvae surviving either on Rad21 or Rad21TEV were prepared before (TEV off) and at various times after shifting third-instar larvae to 30°C (TEV on). Polytene chromosomes were coimmunostained with antibodies against Rad21 and EcR-B1. The chromosome morphology was visualized by DAPI staining. In the overlay (right panels), DAPI is shown in blue, Rad21 in green, and EcR-B1 in red. Scale bars are 50 μm (top two rows) and 100 μm (bottom three rows).
Current Biology  , DOI: ( /j.cub )
Copyright © 2010 Elsevier Ltd Terms and Conditions

6. Figure 4
Loss of Cohesin in Salivary Glands Causes Both Rapid and Slow Changes in Transcript Levels
The kinetics of transcriptional changes upon cleavage of cohesin in salivary glands were assessed by qRT-PCR analysis using the SG-TEV/tubGal80ts (black lines) and hs-TEV (gray lines) systems. Plots show tubulin-normalized, averaged fold differences in transcript levels in the absence (Rad21TEV) versus presence (Rad21) of cohesin over time (in hours; t = −1 indicates time point before TEV induction), obtained from three independent time courses per TEV-expression system (error bars show standard deviations). Genes were classified as early and late response genes based on rapid or gradual changes in transcript levels within the first 4 or 16 hr, respectively. Green indicates upregulation, red indicates downregulation, gray indicates no change in the absence of cohesin.
Current Biology  , DOI: ( /j.cub )
Copyright © 2010 Elsevier Ltd Terms and Conditions

7. Figure 5 Cohesin Is Required for Puffing at Eip74EF and Eip75B
The ecdysone-induced late third-instar puffing response at the neighboring chromosomal bands 74 and 75 (harboring Eip74EF and Eip75B, respectively) was analyzed by polytene chromosome spreads before and after salivary gland-specific TEV protease induction in SG-TEV/tubGal80ts late third-instar larvae surviving either on Rad21TEV or Rad21.
(A) Representative polytene chromosome spreads from salivary glands in the presence (Rad218h and Rad21TEVbefore) and absence of cohesin (Rad21TEV, later time points) were coimmunostained with antibodies against Rad21 and EcR-B1. The chromosome morphology was visualized by DAPI staining. Positions of bands 74 and 75 are indicated in overlays (DAPI in blue, EcR-B1 in red). The two adjacent sharp EcR-stained bands at the right side of band 75 (toward the chromocenter) were used to identify the locus. Scale bars are 50 μm.
(B) Quantification of normalized puff sizes (see Experimental Procedures) at bands 74 and 75 in the presence of cohesin (blue squares and light blue diamonds) and at 2 hr (purple triangles) and 4.5 hr (pink crosses) after cohesin cleavage. Each data point represents the normalized puff size at band 74 plotted against the normalized puff size at band 75 from the same polytene chromosome spread.
(C) Changes in puffing status at bands 74 and 75 upon cleavage of cohesin. Based on the ratio and absolute sizes of the 74 and 75 puffs, each twin locus was classified as either puffed-puffed (P-P, blue), with both 74 and 75 decondensed, unpuffed-puffed (U-P, purple), with 74 condensed and 75 decondensed, or unpuffed-unpuffed (U-U, pink; puffed loci: normalized width > 1.5). The graph plots the percentage of spreads belonging to each category for each experimental condition.
(D) Scheme illustrating the distinct puffing stages at observed before and after TEV cleavage of cohesin. EcR binding to 74 and 75, as well as to the two adjacent bands toward the chromocenter, is highlighted in red.
Current Biology  , DOI: ( /j.cub )
Copyright © 2010 Elsevier Ltd Terms and Conditions